Comparing the cumulative live birth rate of cleavage-stage versus blastocyst-stage embryo transfers between IVF cycles:a study protocol for a multicentre randomised controlled superiority trial (the ToF trial)

Introduction In vitro fertilisation (IVF) has evolved as an intervention of choice to help couples with infertility to conceive. In the last decade, a strategy change in the day of embryo transfer has been developed. Many IVF centres choose nowadays to transfer at later stages of embryo development, for example, transferring embryos at blastocyst stage instead of cleavage stage. However, it still is not known which embryo transfer policy in IVF is more efficient in terms of cumulative live birth rate (cLBR), following a fresh and the subsequent frozen-thawed transfers after one oocyte retrieval. Furthermore, studies reporting on obstetric and neonatal outcomes from both transfer policies are limited. Methods and analysis We have set up a multicentre randomised superiority trial in the Netherlands, named the Three or Fivetrial. We plan to include 1200 women with an indication for IVF with at least four embryos available on day 2 after the oocyte retrieval. Women are randomly allocated to either (1) control group: embryo transfer on day 3 and cryopreservation of supernumerary good-quality embryos on day 3 or 4, or (2) intervention group: embryo transfer on day 5 and cryopreservation of supernumerary good-quality embryos on day 5 or 6. The primary outcome is the cLBR per oocyte retrieval. Secondary outcomes include LBR following fresh transfer, multiple pregnancy rate and time until pregnancy leading a live birth. We will also assess the obstetric and neonatal outcomes, costs and patients' treatment burden. Ethics and dissemination The study protocol has been approved by the Central Committee on Research involving Human Subjects in the Netherlands in June 2018 (CCMO NL 64060.000.18). The results of this trial will be submitted for publication in international peer-reviewed and in open access journals. Trial registration number Netherlands Trial Register (NL 6857)

Stronger T-Cell Alloreactivity and Diminished Suppressive Capacity of Peripheral Regulatory T Cells in Infertile Women Undergoing In Vitro Fertilization

ProblemIncreasing evidence suggests modulation of the maternal immune response to be essential for successful pregnancy. We studied the immunophenotypic profile and function of peripheral blood T lymphocytes in infertile women undergoing in vitro fertilization (IVF) and fertile control population. MethodWe collected peripheral blood mononuclear cells (PBMC) from infertile patients with recurrent implantation failure (RIF), infertile patients with successful IVF (IVFs), and normal fertile women. Cells were phenotypically analyzed, and the proliferative response and cytokine production were studied in mixed lymphocyte cultures (MLC), using lymphocytes of the own partner, or a third-party male as stimulators cells. To examine the suppressive capacity of regulatory regulatory T cells (Tregs), we performed MLC studies with a CD45(+) fraction depleted for CD4(+)CD25(bright) T cells. ResultsNo significant differences in proportions of subsets of circulating T lymphocytes were observed. The proliferative allo-immune response of PBMC of IVF women (RIF and IVFs) was significantly higher, with higher production of T-helper cells (Th1) and Th2 cytokines, compared to the fertile women. This difference in proliferation and cytokine production was associated with a diminished suppressive capacity of Tregs in these women. ConclusionThe higher allo-immune response of the IVF women compared to fertile women might be the result of a diminished suppressive capacity of Tregs and emphasizes the important role of Tregs during conception

Human Extravillous Trophoblasts Penetrate Decidual Veins and Lymphatics before Remodeling Spiral Arteries during Early Pregnancy

<div><p>In humans, the defective invasion of the maternal endometrium by fetal extravillous trophoblasts (EVTs) can lead to insufficient perfusion of the placenta, resulting in pregnancy complications that can put both mother and baby at risk. To study the invasion of maternal endometrium between (W)5.5–12 weeks of gestation by EVTs, we combined fluorescence in situ hybridization, immunofluorescence and immunohistochemistry to determine the presence of (male) EVTs in the vasculature of the maternal decidua. We observed that interstitial mononuclear EVTs directly entered decidual veins and lymphatics from W5.5. This invasion of decidual veins and lymphatics occurred long before endovascular EVTs remodelled decidual spiral arteries. This unexpected early entrance of interstitial mononuclear EVTs in the maternal circulation does not seem to contribute to the materno-placental vascular connection directly, but rather to establish (and expand) the materno-fetal interface through an alternative vascular route.</p></div

Human EVTs penetrate decidual veins early during first trimester (W5.5-W7.5).

<p>(A) Histological sections of decidua basalis at W5.5 used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX/chrY (right) was performed in the pKRT/PECAM1-stained sections. White arrows indicate male EVTs invading veins. The bottom row shows magnifications of the dashed boxes in the top row. (B) Consecutive sections of W5.5 decidua basalis were immunostained for pKRT/PECAM1. (C) FISH for chrX and chrY (right panels) magnifications are shown for the section in dashed box. White arrows point to male EVTs penetrating a decidual vein. (D) Histological sections of decidua basalis at W7.2 used for Azan staining (left panel), immunostained for pKRT and PECAM1 (left-middle panel). FISH for chrX/chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The most right panel shows a magnification of the dashed box. White arrows indicate male EVTs invading veins. (E) Percentage of invaded decidual veins per total veins encountered per histological section (n) between W5.5-W12. Results are sown as mean ± standard deviation. All scale bars are depicted.</p

Human EVTs in the vicinity of arteries early during first trimester (W5.5).

<p>Histological sections of W5.5 decidua basalis showing decidual arteries. Consecutive sections were used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX and chrY (right panel) was performed in the pKRT/PECAM1-stained sections. The (middle and) bottom rows show magnifications of the (numbered) dashed boxes in the top rows. White arrows depict male EVTs. All scale bars are depicted.</p

Human extravillous trophoblast cells invade decidual veins and lymphatics early during first trimester.

<p>(A) Histological sections of decidua basalis from a (mosaic) Klinefelter syndrome (47,XXY and 46, XY) pregnancy at W8.4. Left panels show low magnifications of same section immunostained for pKRT and PECAM1 (top) and chrX/chrY FISH (bottom). Middle-left and middle-right panels are magnifications of the (numbered) dashed boxes in the left panels. Right panels are magnifications of the dashed boxes in the middle-right panels. White arrowheads point to XXY fetal EVTs. (B) Histological sections of decidua basalis at W7.2, W8.4 and W10 immunostained for pKRT, PDPN and PECAM1 (left panels). FISH for chrX and chrY (right) was performed in the in the pKRT/PDPN/PECAM1-stained sections. White arrows point to EVTs invading the lymphatic vessels. All scale bars are depicted.</p

Piwi Proteins and piRNAs in Mammalian Oocytes and Early Embryos

Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi and piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, whereas those in bovine ovary only express PIWIL1. In human, macaque, and bovine ovaries, we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis, we show that these maturing oocytes strongly and specifically express the PIWIL3 protein, alongside other, known piRNA-pathway components. A piRNA pool is still present in early bovine embryos, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal that there are highly dynamic piRNA pathways in mammalian oocytes and early embryos

Human extravillous trophoblast cells in decidua between W8-W12.

<p>(A) Histological sections of decidua basalis at W8.4 used for Azan staining (left panel), immunostained for pKRT and PECAM1 (left-middle panel). FISH for chrX/chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The most right panel shows a magnification of the dashed box. White arrows indicate male EVTs invading veins. (B) Histological sections of decidua basalis at W10 used for Azan staining (left panel), immunostained for pKRT and ACTA2 (left-middle panel) and immunostained for pKRT and PECAM1 (right-middle panel). FISH for chrX/chrY (right) was performed in the pKRT/PECAM1-stained sections. White arrows indicate male EVTs. Low magnifications are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169849#pone.0169849.s002" target="_blank">S2 Fig</a>. (C) Histological sections of W10 and W12 decidua basalis showing remodeled and unremodelled arteries. Consecutive sections were immunostained for pKRT and ACTA2 (left panels) and immunostained for pKRT and PECAM1 (middle panels). FISH for chrX and chrY (right panels) was performed in the pKRT/PECAM1-stained sections. The bottom rows show magnifications of the dashed boxes in the top rows. All scale bars are depicted.</p

Dynamic in vitro culture of cryopreserved-thawed human ovarian cortical tissue using a microfluidics platform does not improve early folliculogenesis

Current strategies for fertility preservation include the cryopreservation of embryos, mature oocytes or ovarian cortical tissue for autologous transplantation. However, not all patients that could benefit from fertility preservation can use the currently available technology. In this regard, obtaining functional mature oocytes from ovarian cortical tissue in vitro would represent a major breakthrough in fertility preservation as well as in human medically assisted reproduction. In this study, we have used a microfluidics platform to culture cryopreserved-thawed human cortical tissue for a period of 8 days and evaluated the effect of two different flow rates in follicular activation and growth. The results showed that this dynamic system supported follicular development up to the secondary stage within 8 days, albeit with low efficiency. Surprisingly, the stromal cells in the ovarian cortical tissue were highly sensitive to flow and showed high levels of apoptosis when cultured under high flow rate. Moreover, after 8 days in culture, the stromal compartment showed increase levels of collagen deposition, in particular in static culture. Although microfluidics dynamic platforms have great potential to simulate tissue-level physiology, this system still needs optimization to meet the requirements for an efficient in vitro early follicular growth

Editorial comment: Ovarian Stimulation for In Vitro Fertilization and Long-term Risk of Breast Cancer (JAMA 2016;316:300–312)

Previous studies have shown that both exogenous and endogenous estrogens and progestogens increase the risk of breast cancer. it has been suggested that in vitro fertilization (IVF) procedures may increase breast cancer risk. This risk could be could be due to a temporary decrease in estradiol and progesterone levels (during down-regulation of the natural cycle), as well as strongly elevated levels (during stimulation phase). Results of previous studies investigating breast cancer risk after IVF treatment were inconclusive because of limited follow-up. Recent studies have reported no increased risk of this cancer after IVT at a mean follow-up of 8 and 16 years. This historical cohort (OMEGA) study quantified long-term risk of breast cancer after ovarian stimulation for IVF. The aim of the study was to compare the long-term risk of breast cancer among a nationwide cohort of women receiving ovarian stimulation for IVF with that of women receiving other fertility treatments and those in the general population. The study was conducted in 12 IVF clinics in the Netherlands. The cohort was composed of 19,158 women who started IVF treatment between 1983 and 1995 (IVF group) and 5950 women who started other fertility treatments between 1980 and 1995 (non-IVF group) Data were obtained from all 12 IVF clinics. There was complete follow-up through December 2013 for 96% of the cohort. At end of follow-up, the median age was 53.8 years in the IVF group and 55.3 years in the non-IVF group. Information on ovarian stimulation for IVF, other fertility treatments, and potential confounders was obtained from medical records and mailed questionnaires. First invasive and in situ breast cancer incidence among women who underwent fertility treatments was obtained through linkage with the Netherlands Cancer Registry (1989-2013). Risk of breast cancer in the IVF group was compared with risk in the non-IVF group (hazard ratios [HRs]) and the general population (standardized incidence ratios [SIRs]). In the total cohort, the mean age at baseline was 32.8 years, and the mean number of IVF cycles was 3.6. After a median follow-up of 21.1 years, 839 of the cohort had invasive breast cancer, and 109 had in situ breast cancer. Compared with the general population, breast cancer risks were not increased either in the IVF group (SIR, 1.01; 95% confidence interval [CI], 0.93-1.09) or the non-IVF group (SIR, 1.00; 95% CI, 0.88-1.15). At the age of 55 years, cumulative incidence rates of breast cancer were 3.0% in the IVF group and 2.9% in the non-IVF group (P = 0.85). With longer time since treatment (>= 20 years), the SIR did not increase in the IVF group (SIR, 0.92; 95% CI, 0.73-1.15) or in the non-IVF group (SIR, 1.03; 95% CI, 0.82-1.29). Women with 7 or more IVF cycles had a significantly decreased risk of breast cancer than did women who were treated with 1 to 2 IVF cycles; the HR was 0.55, with a 95% CI of 0.39 to 0.77. The risk was also significantly lower (HR, 0.77; 95% CI, 0.61-0.96) after poor response to the first IVF cycle (<4 collected oocytes) versus normal response (>= 4 collected oocytes). These data show that IVT treatment did not increase the risk of breast cancer in a cohort of women undergoing fertility treatment in the Netherlands between 1980 and 1995 when compared with women who received non-IVF treatment or those in the general population after a median follow-up of 21 years. These findings are consistent with those from recent reviews showing no significant increase in long-term risk of breast cancer among IVF-treated women